Chromatograms - Qualitative and Quantitative Analysis

Chromatograms - Qualitative and Quantitative Analysis

• Chromatograms can be used for both qualitative and quantitative analysis.
• Quantitative analysis methods include Internal Normalisation, External Standard, and the Use of Internal Standards.
• Qualitative analysis is useful for identifying analytes by their retention time.

Qualitative Analysis

• Retention time (t_R) remains relatively constant for a compound under the same conditions and equipment.
• This consistency aids in analyte identification.

Quantitative Analysis

• Different techniques are used for GC and HPLC.
• Three main methods for GC and HPLC quantitative analysis are:
  • Internal Normalisation
  • Use of External Standard
  • Use of Internal Standard.

Quantitative Analysis in GC - Internal Normalisation

• If a compound's amount gives the same peak area, internal normalisation calculates the percentage of each component in a mixture.
• Example: % Component X = (Area of Component X Peak / Total Area of All Peaks) x 100
• Detector responses may differ for compounds. External calibration or internal standard methods are then used to account for such differences.

External Standard

• A series of samples (each with a known amount of test substance) are subjected to identical GC conditions.
• A calibration curve (response area vs. concentration) is created.
• The concentration of a test sample is determined from the calibration curve and its measured response.

External Standard - Example Question

• An analyst determines aspirin in tablet extract via GC.
• Known aspirin concentrations in methanol are injected and peak areas recorded.
• A calibration graph (aspirin concentration vs. peak area) is plotted.
• An extract sample with an aspirin peak area of 0.35cm² is analyzed using the calibration graph to find its concentration.

Internal Standard

• This approach overcomes the difficulties of external calibration.
• An internal standard is added to the sample to be analyzed.
  • This is called "spiking"
• The ratio of peak areas between a sample's analyte and the internal standard is constant regardless of variation during analysis.

Internal Standard - Requirements

• An internal standard must:
  • Be closely related to the assayed component
  • Be completely resolved from the assay component and all other components
  • Have a close retention time to the compound being analyzed
  • Be used at a concentration similar to the expected concentration of the analyte in the sample

BP Format for Assays Requiring Internal Standards

• Three samples are required:
  • Calibration standard: Contains approximate equal amounts of the pure standard and internal standard.
  • Extract from the sample with no internal standard (for validating).
  • Extract from the sample with the same internal standard as in solution 1 (sample to be analyzed).

Solution 1: Standard Solution (Calibration Solution)

• Detectors do not always respond equally to the same amount of different compounds.
• A response factor (Rf) is calculated to determine the ratio of analyte response vs. internal standard in the sample.

Solution 3: Unknown Solution (Sample Solution Spiked With IS)

• A known concentration of internal standard is added to a sample before analysis.
• The ratio of analyte area to internal standard area is the Response Factor (Rf) for that unknown sample.
• Combining data allows calculation of the analyte concentration in an unknown mixture spiked with internal standard.

Advantages of Internal Standard

• Compensates for changes in chromatographic conditions when both standards and the internal standard are affected equally.
• Is unaffected by variations in injection volume.

Applications of GC in Biomedical and Pharmaceutical Analysis

• Drug and metabolite measurement in biological fluids.
• Unformulated drug characterization/process impurity detection.
• Limit test for solvent residues, etc, in drug substances.
• Drug quantification in formulations.
• Characterization of raw materials and volatile/proprietary mixtures.

Strengths of GC

• Capable of high precision during quantitative analysis.
• Advances in technology and greater separating power when used with capillary columns.
• Readily automated.
• Can use with non-volatile compounds
• Using helium is cost-effective.

Weaknesses of GC

• Analysis is limited to only stable, volatile compounds.
• Samples may require derivatization.
• Injecting small volumes can be difficult.
• Aqueous solutions and salts cannot be analyzed.

Applications of HPLC in Biomedical and Pharmaceutical Analysis

• Used for quantitative analysis of pharmaceutical products and is the industry standard method.
• Drug stability monitoring in formulations.
• Determining partition coefficients and pKa values of drugs, along with protein binding.

Strengths of HPLC

• Precise sample introduction, ensuring quantitative accuracy.
• Rapid technology advancements.
• Varied column, stationary phase, and mobile phase options, with broad applicability.
• Lower risk of sample degradation.
• Ready automation.

Weaknesses of HPLC

• Requires a potentially expensive detector to analyze compounds without chromophores.
• Generates a significant amount of organic solvent waste.
• Prior extraction of drugs from formulations is often necessary.

Explore the critical concepts behind chromatograms, focusing on both qualitative and quantitative analysis methods. Learn about retention time, internal normalisation, and the differences between techniques used in GC and HPLC. This quiz will enhance your understanding of how these methods aid in analyte identification and quantification.